Usefulness of administration of non-organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using paraoxon.

Reversible acetylcholinesterase (AChE) inhibitors can protect against the lethal effects of irreversible organophosphorus AChE inhibitors (OPCs), when administered before OPC exposure. We have assessed in vivo the mortality-reducing efficacy of a group of known AChE inhibitors, when given in equitoxic dosage before exposure to the OPC paraoxon. Protection was quantified in rats by determining the relative risk (RR) of death. Best in vivo protection from paraoxon-induced mortality was observed after prophylactic administration of physostigmine (RR = 0.30) or the oxime K-27 (RR = 0.34); both treatments were significantly superior to the pre-treatment with all other tested compounds, including the established substance pyridostigmine. Tacrine (RR = 0.67), ranitidine (RR = 0.72), pyridostigmine (RR = 0.76), tiapride (RR = 0.80) and 7-MEOTA (RR = 0.86) also significantly reduced the relative risk of paraoxon-induced death, but to a lesser degree. Methylene blue, amiloride and metoclopramide had an unfavorable effect (RR ≥ 1), significantly increasing mortality. When CNS penetration by prophylactic is undesirable K-27 is a promising alternative to pyridostigmine.

Impact of paraoxon followed by acetylcholinesterase reactivator HI-6 on gastric myoelectric activity in experimental pigs.

OBJECTIVES: Organophosphorus compounds represent nerve agents, pesticides and several industrial compounds. Treatment after exposure to organophosphates involves the use of parasympatolytics, acetylcholinesterase (AChE) reactivators/modulators and anticonvulsive drugs. Wider clinical use of several AChE reactivators/modulators might be limited because of possible side effects, including gastrointestinal toxicity. In this study we evaluated the effect of paraoxon and an AChE reactivator (HI-6) on the gastric myoelectric activity in experimental pigs. METHODS: Six female experimental pigs (mean weight 33 kg) entered the study. Intramuscular paraoxon (1.5 g) was administrated after the baseline gastric electrogastrography (EGG) recording, followed by HI-6 dimethansulphonate (1.5 g i.m.) 10 min. later. A further ten 15-minute-interval EGG recordings were performed. Running spectral analysis was used for the elemental evaluation of the EGG. The results were expressed as dominant frequency of slow waves at all intervals of EGG recordings. EGG power analysis was performed in all animals. RESULTS: Paraoxon induced a non-significant decrease of dominant frequency (2.8±0.6 vs. 2.6±0.5 cycles per min.; p=0.092). Subsequent administration of HI-6 normalised dominant frequency to basal values and increased it significantly within the subsequent 30 minutes (3.0±0.4; p<0.001). Paraoxon administration did not influence the power (within a 10-minute exposure). However, the amplitudes increased significantly 90 minutes after administration of HI-6 (819±109 vs. 5054±732 µV2; p<0.001). CONCLUSIONS: AChE reactivator HI-6 blocked the gastric effect of paraoxon significantly. Subsequent myoelectric changes in the dominant frequency and power were executed by HI-6. The effect of paraoxon was non-significant.

Nano-intercalated organophosphorus-hydrolyzing enzymes in organophosphorus antagonism.

A dendritic poly(2-alkyloxazoline)-based polymer was studied as a new carrier system for the organophosphorus-hydrolyzing recombinant enzymes, organophosphorus acid anhydrolase and organophosphorus hydrolase. Paraoxon (PO) and diisopropylfluorophosphate (DFP) were used as model organophosphorus compounds. Changes in plasma cholinesterase activity were monitored. The cholinesterase activity was proportional to the concentrations of DFP or PO. Plasma cholinesterase activity was higher in animals receiving enzyme and oxime before the organophosphates than in the oxime-only pretreated groups. These studies suggest that cholinesterase activity can serve as an indicator for the in vivo protection by the nano-intercalated organophosphorus acid anhydrolase or organophosphorus hydrolase against organophosphorus intoxications. These studies represent a practical application of polymeric nano-delivery systems as enzyme carriers in drug antidotal therapy.

Balanced modulation of neuromuscular synaptic transmission via M1 and M2 muscarinic receptors during inhibition of cholinesterases.

Organophosphorus (OP) compounds that inhibit acetylcholinesterase are a common cause of poisoning worldwide, resulting in several hundred thousand deaths each year. The pathways activated during OP compound poisoning via overstimulation of muscarinic acetylcholine receptors (mAChRs) play a decisive role in toxidrome. The antidotal therapy includes atropine, which is a nonspecific blocker of all mAChR subtypes. Atropine is efficient for mitigating depression in respiratory control centers but does not benefit patients with OP-induced skeletal muscle weakness. By using an ex vivo model of OP-induced muscle weakness, we studied the effects of the M1/M4 mAChR antagonist pirenzepine and the M2/M4 mAChR antagonist methoctramine on the force of mouse diaphragm muscle contraction. It was shown that weakness caused by the application of paraoxon can be significantly prevented by methoctramine (1 µM). However, neither pirenzepine (0.1 µM) nor atropine (1 µM) was able to prevent muscle weakness. Moreover, the application of pirenzepine significantly reduced the positive effect of methoctramine. Thus, balanced modulation of neuromuscular synaptic transmission via M1 and M2 mAChRs contributes to paraoxon-induced muscle weakness. It was shown that methoctramine (10 µmol/kg, i.p.) and atropine (50 µmol/kg, i.p.) were equieffective toward increasing the survival of mice poisoned with a 2xLD50 dose of paraoxon.

Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system.

Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and "ongoing inhibition") of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and the model applied and may be considered an external validation. The most sensitive component (Eα) for paraoxon (11-23% of activity, I(50) (30 min)=9-11 nM) is also the most sensitive for mipafox (I(50) (30 min)=4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (Eß, 71-84% of activity; I(50) (30 min)=1216 nM) is practically resistant to mipafox. The third component (Eγ, 5-8% of activity) is paraoxon resistant and has I(50) (30 min) of 3.4 µM with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies.

Carbon nanotube-polymer based nanocomposite as electrode material for the detection of paraoxon.

Biosensor based on the inhibition of enzymes has been used for the detection of organophosphorous compounds wherein amperometic method has been employed. Carbon nanotubes (CNT) has been grown over YNi3 alloy hydrides and purified for further use. The high surface area and the acidic sites created during the purification of CNT with oxidizing acids have been exploited for the adsorption and entrapment of the enzyme acetylcholine esterase. In the present work, conducting polymer polypyrrole has been uniformly coated over the CNT surface using chemical oxidative technique. The nanocomposite was characterized by scanning electron microscopy (SEM) and High resolution transmission electron microscopy (HRTEM). In the present report high catalytic activity of CNT towards the electroxidation of thiocholine has been utilized for the detection of organophosphorous compound paraoxon. Developed biosensor uses the principal of acetylcholinesterase inhibition by nerve agent and hence reduction in oxidation current of thiocholine for the detection of paraoxon. Synthesized PPY-MWNT nanocomposite has been used for the electrode preparation over GC electrode. Due to high porosity of polymer and high electrical conductivity of CNT, a detection level of 3 nM paraoxon could be achieved. The details of fabrication of the sensor and the dependence of the sensitivity have been discussed.

Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats.

We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 microl/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at <0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed these concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 microM, 1.5 microl/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 microM and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 microM paraoxon but was transiently elevated (0.5-1.5 h) with 10 microM paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very different views into enzyme-inhibitor interactions. Furthermore, the proliferation/migration of cells containing high amounts of cholinesterase just adjacent to a dialysis probe could affect the recovery and thus detection of extracellular acetylcholine in microdialysis studies.

Paraoxon: effects on rat brain cholinesterase and on growth hormone and prolactin of pituitary.

Cholinesterase activity of brain and content of growth hormone and prolactin in the pituitary were compared after short-term (3 days) and long-term (14 days) treatment with paraoxon in male and female rats. Within 3 days cholinesterase activity was reduced to between 5 and 15 percent of that in controls. The content of growth hormone in the pituitary was increased in long-term experiments by 50 percent. This increase in paraoxon-treated animals-suggests a possible role of a cholinergic mechanism in the regulation of growth hormone secretion.

Type of cell death and the role of acetylcholinesterase activity in neurotoxicity induced by paraoxon in cultured rat hippocampal neurons.

Organophosphate (Ops) neurotoxicity is attributed both to its well-known cholinergic and non-cholinergic effects. In the present study we compared enzymatic and morphologic changes in neurons exposed to paraoxon during one day and one week. The effect of exposure time is important in neurotoxicity of Ops. The longer the exposure time is the more damage is observed in neurons, although there are few investigations about the effect in the post-exposure period. Hippocampal cells were obtained from rat neonates and cultured in Neurobasal/B27. Paraoxon at 50 and 100 microM were added. Inverted microscope and electron microscope were used to study cell morphology and Neutral Red staining was used to measure viability. We also assayed caspase-3 and (acetylcholinesterase) AChE activity. Hoechst staining was utilized to determine the type of cell death. Culture medium was replaced after 24 h in one-day group, however, tests were all carried out at the end of the first week in both group. The results indicate that paraoxon reduced the viability in a dose-dependent manner. Our results do not confirm apoptosis in either group; it seems that the cell death in one-day exposure group was not AChE dependent. In conclusion, present data imply that the toxicity of paraoxon is both dose and duration dependent, which may even remain after the cessation of exposure.

Mechanisms of action of paraoxon, an organophosphorus pesticide, on in vivo dopamine release in conscious and freely moving rats.

Paraoxon is the active metabolite of parathion, an organophosphorus pesticide which can cause neurotoxic effects in animals and humans. In the present work, we investigated the effects of 5 mM paraoxon on striatal dopamine, DOPAC and HVA levels in conscious and freely moving rats, after treatment with TTX, reserpine, nomifensine, KCl, Ca++-free/EDTA medium, AP-5 or L-NAME. The intrastriatal administration of paraoxon for 60 min, through the microdialysis probe, significantly produced an increase of the dopamine to 1066 ±â€¯120%, relative to basal levels. Administration of paraoxon to 20 µM TTX, 10 mg/kg reserpine or Ca++-free/EDTA medium-pretreated animals decreased the dopamine levels to 73%, 81%, and 70%, respectively, when compared with the effect of 5 mM paraoxon. Infusion of 50 µM nomifensine induced a maximal increase in extracellular dopamine levels to 1435 ±â€¯387%, and when nomifensine was coadministered with paraoxon, striatal dopamine levels increased to 2429 ±â€¯417%, an increase that was ∼230% higher that observed with the administration of the pesticide alone. Coinfusion of KCl and paraoxon produced an increase in extracellular dopamine to 1957 ±â€¯445%, that was significantly higher than that observed with POX or KCl (1104 ±â€¯220%) administered individually. Pretreatment with 650 µM AP-5 or 100 L-NAME reduced the effect of paraoxon on extracellular dopamine levels by 49.1% and 53.7%, respectively. Our results suggest that paraoxon induces dopamine release by a vesicular-, Ca++-, and deporalization-dependent mechanism, being independent of dopamine transporter. In addition, the paraoxon-induced dopamine release is mediated by glutamatergic and nitrergic neurotransmitter systems.

Leptin decreases plasma paraoxonase 1 (PON1) activity and induces oxidative stress: the possible novel mechanism for proatherogenic effect of chronic hyperleptinemia.

Obesity is an important risk factor of atherosclerosis; however, the mechanism of proatherogenic effect of obesity is not definitely established. Recent studies suggest an important role of leptin in obesity associated complications. We investigated the effect of chronic hyperleptinemia on two antioxidant enzymes contained in plasma lipoproteins: paraoxonase 1 (PON1) and platelet activating factor-acetylhydrolase (PAF-AH). The study was performed on three groups of male Wistar rats: (1) control, fed ad libitum, (2) leptin treated, receiving leptin (0.25 mg/kg twice daily s.c. for 7 days), (3) pair-fed, in which food intake was identical as in leptin-treated animals. PON1 activity toward paraoxon, phenyl acetate, gamma-decanolactone and homogentisic acid lactone was lower in leptin-treated than in control group by 30.4, 30.8, 34.5 and 62%, respectively. Leptin increased plasma concentration and urinary excretion of isoprostanes by 46.4 and 49.2%, respectively. Leptin treatment had no effect on plasma lipid profile and glucose level. Plasma leptin was 208.8% higher in leptin-treated and 51.5% lower in pair-fed than in control group. These data indicate that hyperleptinemia induced by exogenous leptin administration markedly decreases plasma PON1 activity and induces oxidative stress. These mechanisms may be involved in atherogenesis in hyperleptinemic obese individuals.

Carboxylesterases in guinea pig. A comparison of the different isoenzymes with regard to inhibition by organophosphorus compounds in vivo and in vitro.

The different isoenzymes of carboxylesterase (CarbE) from guinea pig liver, lung and plasma were separated by gel filtration and chromatofocusing. The isoenzymes were characterized by inhibition with several different organophosphorus compounds. The bimolecular rate constants showed the same tendency of decreased inhibition for all of the isoenzymes in the order; paraoxon greater than soman greater than diisopropylphosphofluoridate (DFP) greater than bis(p-nitrophenyl)phosphate. With two exceptions the inhibition constants for the different isoenzymes differed little. Subcutaneous and intraperitoneal administration of DFP and paraoxon rapidly inhibited the CarbE activity in guinea pig plasma. Much higher doses were necessary to obtain a marked inhibition in lung and liver. About 25% of CarbE activity in lung was resistant to paraoxon and DFP inhibition. Gel filtration of lung homogenate after treatment with the organophosphorus compounds showed that the CarbE activity of the medium molecular mass fractions was inhibited only weakly. This could be due to reduced accessibility to some of the lung CarbE isoenzymes.

Central effects of paraoxon on haemodynamics in the cat.

Application of paraoxon into the left vertebral artery (8--80 micrograms) or both the left and right vertebral artery (4--8 micrograms) of the anaesthetized cat evoked dose-dependent depressor effects, whereas heart rate was not influenced significantly. Also after systemic administration of paraoxon (150--825 micrograms . kg-1), while peripheral muscarinic receptors were blocked, depressor effects were still observed. Dose-response curves for the depressor response to paraoxon were established. Infusion of low doses of dexetimide via the vertebral artery prevented the hypotensive action of paraoxon. The distribution of this antimuscarinic drug in the brain was investigated. The depressor effect of paraoxon can be attributed to both a decrease in peripheral resistance and cardiac output. Decerebration and midcollicular transection were carried out in order to elucidate the site and mechanism of action. The depressor effect of paraoxon seems to be mediated by a central mechanism of action located within the lower brain stem. It is concluded that stimulation of muscarinic receptors in the pontomedullary region gives rise to the observed changes in haemodynamic parameters. Muscarinic receptors in the hypothalamus seem to be of minor importance for the hypotensive action of paraoxon.

Neurotoxicity of acute and repeated treatments of tabun, paraoxon, diisopropyl fluorophosphate and isofenphos to the hen.

The neuropathic potential of acute and repeated exposures of the phosphoramidates tabun (GA) and isofenphos (IFP), of diisopropyl fluorophosphate (DFP) and paraoxon (PO) were examined in the hen with treatments for up to 90 days via intramuscular injections of the highest tolerated doses with atropine protection. Plasma acetylcholinesterase (AChE), non-specific butyrylcholinesterase (BChE) and creatine kinase (CK) activities were measured in order to monitor whether the compounds were present at biologically active concentrations. Locomotor behavior was observed and tissues from the peripheral and central nervous systems were examined for signs of organophosphate-induced delayed neuropathy (OPIDN). No behavioral or histological evidence of OPIDN was observed after treatments with GA, IFP, PO, saline or atropine sulfate. DFP-treated birds displayed locomotor and neuropathological signs of OPIDN with a no effect level (NOEL) between 25 and 50 micrograms/kg.

Spine density and dendritic branching pattern of hippocampal CA1 pyramidal neurons in neonatal rats chronically exposed to the organophosphate paraoxon.

The organophosphate cholinesterase (ChE) inhibitor paraoxon is the oxidized active metabolite of parathion, a pesticide whose use in agriculture has been matter of increasing concern. The present work was aimed at reproducing a prolonged exposure to low concentrations of paraoxon and assessing possible damage to the hippocampus during the period of most significant cholinergic development. Male Wistar rats were given, from P8 to P20, subcutaneous daily injections of paraoxon (0.1, 0.15 and 0.2mg/kg). The rate of body weight gain was reduced by all doses of paraoxon and brain ChE activity progressively decreased up to 60% by P21. Some deaths occurred in the beginning of the treatment, but the surviving animals showed neither convulsions nor overt signs of cholinergic hyperstimulation. Morphometric analysis of Lucifer Yellow-stained CA1 pyramidal neurons in coronal sections of the hippocampus showed that by P21 paraoxon caused a decrease in spine density on basal but not on secondary apical dendrites. The dendritic arborization and the pyramidal and granular cell body layers were not altered by paraoxon. ChE staining decreased in all hippocampal and dentate gyrus regions studied, whereas choline acetyltransferase (ChAT) and zinc-positive fibers remained as in control. In summary, chronic exposure to low paraoxon concentrations during the period of rapid brain development caused significant and selective decrease in basal dendritic spine density of the CA1 pyramidal neurons. Distinct modulation of the basal tree at the stratum oriens by the interplay of cholinergic afferent and GABAergic interneurons, as well as the remodeling process in response to a repetitive and rather mild paraoxon insult, may account for this selective susceptibility of basal dendritic spines. The hippocampal alterations described here occurred in the absence of toxic cholinergic signs and may affect brain development and cause functional deficits that could continue into adulthood.

Regional brain cholinesterase activity in rats injected intraperitoneally with anatoxin-a(s) or paraoxon.

Adult male Long-Evans rats were injected intraperitoneally with 1.5, 3.0 or 9.0 micrograms/kg of anatoxin-a(s) that had been extracted from laboratory-grown Anabaena flos-aquae NRC-525-17, 800 micrograms/kg of paraoxon, or a control solution. Blood, anterior spinal cord, and brain cerebellar, cortical, medullary, midbrain, hippocampal, hypothalamic, olfactory and striatal cholinesterase activity was determined in rats that died prior to 2 hours or were anesthetized and killed at 2 hours. Unlike paraoxon, anatoxin-a(s) did not cause detectable inhibition of cholinesterase in the central nervous system, but did cause inhibition of cholinesterase in blood, suggesting that anatoxin-a(s) is strictly a peripheral cholinesterase inhibitor.

Acute effects of the organophosphate paraoxon on schedule-controlled behavior and esterase activity in rats: dose-response relationships.

The effects of acute intraperitoneal administration of paraoxon on behavioral and biochemical parameters were studied in male rats. Rats were trained to press a lever under an FR10 schedule of reinforcement. Rats were injected with 3 sublethal doses of paraoxon (0.5, 0.75, and 1.0 mg/kg) and performance was monitored for four days after exposure. Response rates were depressed significantly for days 1 and 2 with 0.75 and 1.0 mg/kg, but not 0.5 mg/kg, even though there was inhibition of brain and plasma cholinesterases at all doses. Performance recovered prior to brain AChE recovery. There was no clear-cut threshold of brain AChE inhibition required to yield performance deficits, nor was there a direct correlation between significant inhibition in peripheral enzymes which could serve as markers (plasma aliesterases, butyrylcholinesterase, non-iso-OMPA-sensitive cholinesterase, and hepatic aliesterases) and performance deficits, suggesting that other noncholinergic targets may play a role in OP-induced behavioral deficits.

Effects of low concentrations of paraoxon on Ca(2+) mobilization in a human parotid salivary cell-line HSY.

The salivary gland is a target organ of organophosphate pesticides (OPs). Inhibition of acetylcholinesterase (AChE) by OPs leads to a decrease in acetylcholine (ACh) breakdown that results in overstimulation of muscarinic cholinergic receptors (mChR). However, OPs may also directly interact with downstream elements of the phosphoinositide (PI) signalling pathway coupled with mChR. The present study examined the effects of exposure to low concentrations of the OP paraoxon on inositol 1,4,5-trisphosphate (IP(3)) formation and Ca(2+) mobilization in response to ACh or ATP in the human parotid cell-line HSY. Exposure to 0.1 and 1 nM, but not 10 nM, paraoxon for 24 hr significantly elevated the basal cytosolic free Ca(2+) ([Ca(2+)](i)). This increase was abolished by atropine. Ca(2+) release from the IP(3)-sensitive store in response to ACh or ATP, a P2Y-nucleotide agonist, was significantly increased in cells pre-exposed to 0.1 nM paraoxon. However, IP(3) formation was inhibited by paraoxon but mChR expression was not altered. Although IP(3) receptor expression was not changed, Ca(2+) release elicited by IP(3) in streptolysin O toxin-permeabilized cells was significantly larger in cells pre-exposed to 0.1 nM paraoxon, suggesting that paraoxon increases the sensitivity of IP(3) receptors. Paraoxon exposure also induced a concentration-dependent reduction in the total capacity of intracellular Ca(2+) stores, whereas the capacity of the IP(3)-sensitive Ca(2+) store was not altered by paraoxon, as judged by discharging of the IP(3)-sensitive Ca(2+) store with thapsigargin (TG). Ca(2+) influx stimulated by ACh or ATP was also enhanced by 0.1 nM, but not 1 and 10 nM, paraoxon. On the other hand, Ca(2+) influx activated by TG was enhanced by exposure to all concentrations of paraoxon, indicating that paraoxon modulates the Ca(2+) entry pathway. These results suggest that low concentrations of paraoxon interact with elements of the PI pathway, enhancing Ca(2+) release and influx mechanisms.

Neurofilament 200 as an indicator of differences between mipafox and paraoxon sensitivity in Sy5Y neuroblastoma cells.

Organophosphorus (OP) compounds produce potent neurotoxic effects in humans, including organophosphorus-induced delayed neuropathy (OPIDN). This investigation examined the potential for the 200-kD neurofilament protein (NF200) and other neuronal proteins to serve as indicators for neurite damage in a differentiated SY5Y human neuroblastoma cell culture system. Mipafox, which induces OPIDN, increased NF200 protein expression in SY5Y cells differentiated with human recombinant beta-nerve growth factor (NGF, 20 ng/ml) in a concentration-dependent manner, compared to NGF controls, when SY5Y cells were exposed to 0.3 or 30 microM mipafox during the last 5 days of neurite extension (experimental set A). However, mipafox produced little change in NF200 protein expression in SY5Y cells exposed continuously throughout neurite elongation (experimental set B). Paraoxon (up to 30 microM), which does not produce OPIDN, did not produce any change in NF200 expression in set A or set B. The upregulation of NF200 by mipafox may represent a compensatory response to neurite degeneration. Two other neuronal proteins, growth-associated protein 43 (GAP43) and microtubule-associated protein 2ab (MAP2ab), showed no changes in response to OP treatment in NGF-treated cells. Protein expression of NF200 was shown to be an indicator by which the sensitivities of SY5Y cells to mipafox and paraoxon were distinguishable at the molecular level. These results indicate an alternative approach and test system for investigating structure-activity relationships of OPs.

Critical neurohistopathologic evaluation of clinically healthy commercial single-comb White Leghorn hens.

A neurohistopathologic study was conducted to determine the types and incidence of spontaneous lesions of the nervous system in healthy adult hens. Tissues examined were brain, three levels of spinal cord, and sciatic nerves from 92 healthy commercial hens from a single commercial egg-production flock judged above average in health. These hens represented negative controls (untreated or given corn oil) in 9 separate toxicologic studies. Minimal to mild focal proliferative or degenerative microscopic changes were detected at all levels of the nervous system. More than 50% of the hens had lymphocytic perivascular cuffing and focal gliosis in the central nervous system. Similarly, lymphocytic foci (in nerve sheaths or within fasciculi) and Schwann cell hyperplasia were commonly seen in sciatic nerves. The spinal cord exhibited neuronal swelling and chromatolysis frequently; axonal degeneration (in brain and spinal cord) and swelling or degeneration of nerve fibers in sciatic nerves were observed sporadically. Most changes were rare or mild. A few chickens had mild or moderately severe perivascular cuffing and focal gliosis. The significance of these findings is discussed in relation to the use of the adult hen in neurotoxicologic evaluations.